Project Abstract Blockade of lymphocyte chemotaxis is a promising strategy for the prevention of graft-versus-host disease (GvHD) following allogeneic hematopoietic stem-cell transplantation (alloHSCT). Specifically, blockade of the chemokine receptor CCR5 has so far been examined in several clinical trials and clinical results appear promising, showing low rates of acute GvHD without an increase in relapse risk or infectious complications. However, prophylaxis failure still occurred in some patients and data in humans and murine suggest that signaling through CXCR3 serves as a resistance mechanism. Dual CCR5/CXCR3 blockade is a potential strategy, but a detailed understanding of chemokine receptor signaling in alloreactive T-cell migration is needed first. We propose to study the individual roles of CCR5 and CXCR3 in T-cell migration into target organs and the impact of potential blocking strategies. The central hypothesis of this proposal is that CCR5 and CXCR3 are complementary pathways in alloreactive T-cell migration and that combined blockade of both receptors will lead to greater protection against GvHD compared to single-receptor blockade. In support of this hypothesis, we show preliminary data that demonstrate distinct functional properties for CCR5-and CXCR3-expressing T- cells in GvHD, evidence for CXCR3 operating as a resistance mechanism to CCR5 blockade and evidence that CCR5 and CXCR3 ligands activate T-cells through STAT3 independent of their effect on T-cell navigation. To test this hypothesis, we will use murine models and clinical data and biospecimens from 167 patients enrolled on 3 clinical trials to examine the relationship between CCR5 and CXCR3 in GvHD. In Aim 1, we will identify the resistance mechanisms to CCR5 blockade in alloHSCT recipients and identify a novel role for conditioning agents in modulating the role of chemokine receptors in GvHD. In Aim 2, we will determine the complementary roles of CCR5 and CXCR3 in tissue-infiltrating alloreactive T-cells using a novel single-cell methodology in mice and humans. This technology will allow us to assess the potential role of additional chemokine receptors and homing molecules. Aim 3 will examine the impact of dual CCR5/CXCR3 blockade approach on the graft-versus-host and graft-versus-leukemia responses in mouse models. This translational study will fuel our mechanistic understanding of T-cell migration in GvHD, and enable the development of a strategy of combined receptor blockade in humans.